Project Abstract Our sense of balance relies on vestibular hair cells, which detect head movements. Mammals have type I and type II vestibular hair cells, which have distinct morphological, molecular, and physiological properties and unique forms of afferent innervation. Only type II hair cells are replaced in adult mammals under normal conditions and after damage. We do not understand the specific functions of type I and II hair cells nor the types of information they encode and send to the brain via the vestibular nerve. Furthermore, little is known about the mechanisms controlling the production and maintenance of each hair cell type. This knowledge is critical for developing effective therapies to restore vestibular function, through cell regeneration, transplantation, or nerve stimulation (vestibular implants). In mature mammals, the transcription factor Sox2 is expressed in type II hair cells but not in type I hair cells. We recently made the exciting discovery that conditional deletion of Sox2 from type II hair cells of adult mice causes them to acquire morphological and molecular features of type I hair cells and to establish calyx innervation typical of type I hair cells. This transdifferentiation resulted in a significantly higher proportion of type I:II hair cells in all zones of the vestibular epithelia. In this proposal, a team of investigators with distinct but highly connected areas of expertise will exploit our new-found ability to induce vestibular hair cell transdifferentiation in mature mammals to advance our understanding of how type I and II hair cells acquire and maintain their unique properties and how each hair cell type contributes to vestibular function. In Aim 1, we will further test if Sox2 loss-of-function in adult type II hair cells causes them to convert into type I hair cells, and we will determine if Sox2 gain-of-function in adult type I hair cells induces their transdifferentiation to type II hair cells. Our ability to alter proportions of type I and II hair cells in adult mice provides an opportunity to address long-standing questions regarding the functional differences between these cell types. In Aim 2, we will assess the functional contributions of each hair cell type to afferent nerve activity and to animal behavior by altering the type I:II ratio via Sox2 conditional knock-out or ectopic expression. The establishment of type I and II specificity relies on a precise pattern of gene expression reflected in the unique chromatin state of each cell type. In Aim 3, we will analyze the state of chromatin of normal type I versus type II hair cells and study how these chromatin states change during transdifferentiation after Sox2 manipulation in adulthood. !